This investigation is directed at investigation of early changes in the synthesis of DNA and its associated proteins during the onset of transformation. We propose to analyze the synthesis of DNA and of proteins binding to DNA during the onset of transformation in cells transformed by thermosensitive transformation mutants of oncogenic viruses. DNA will be examined by CsCl gradient analysis and DNA fiber autoradiography, in order to examine differences in initiation of DNA synthesis in the presence and absence of transformation or serum. We will purify a DNA-binding protein whose synthesis has already been associated with the onset of transformation, in order to test its biological activity in DNA-unwinding and DNA-replication assays in vitro. The SS93 DNA-binding protein will be purified by column chromatography, and its ability to induce DNA unwinding using melting profile analysis and electron microscopic analysis will be tested. The relationship of SS93 synthesis to transformation and serum-stimulation will be investigated in several cell lines transformed by a variety of oncogenic viruses. The ability of SS93 and other cellular protein extracts of transformed cells to influence the replication of DNA in permeabilized cells in vitro will be investigated. The DNA replication in vitro will be analyzed by density gradient centrifugation in neutral and alkaline CsCl. Alterations in the phosphorylation of SS93 and other DNA-binding proteins will be analyzed during the onset of transformation. Results of this project should provide information concerning proteins involved in the control of DNA replication in normal and transformed cells, in the presence and absence of serum factors.